Correction of vasopressin deficit in the lateral septum ameliorates social deficits of mouse autism model.

Intellectual and social disabilities are common comorbidities in adolescents and adults with MAGE family member L2 (MAGEL2) gene deficiency characterizing the Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. The cellular and molecular mechanisms underlying the risk for autism in these syndromes are not understood. We asked whether vasopressin functions are altered by MAGEL2 deficiency and whether a treatment with vasopressin could alleviate the disabilities of social behavior. We used Magel2-knockout mice (adult males) combined with optogenetic or pharmacological tools to characterize disease modifications in the vasopressinergic brain system and monitor its impact on neurophysiological and behavioral functions. We found that the activation of vasopressin neurons and projections in the lateral septum were inappropriate for performing a social habituation/discrimination task. Mechanistically, the lack of vasopressin impeded the deactivation of somatostatin neurons in the lateral septum, which predicted social discrimination deficits. Correction of vasopressin septal content by administration or optogenetic stimulation of projecting axons suppressed the activity of somatostatin neurons and ameliorated social behavior. This preclinical study identified vasopressin in the lateral septum as a key factor in the pathophysiology of Magel2-related neurodevelopmental syndromes.

Neuroanatomical distribution and function of the vasopressin V1B receptor in the rat brain deciphered using specific fluorescent ligands.

It is now accepted that vasopressin, through V1A/V1B receptors, centrally regulates cognitive functions such as memory, affiliation, stress, fear and depression. However, the respective roles of these receptor isoforms and their contribution to stress-related pathologies remain uncertain. The development of new therapeutic treatments requires a precise knowledge of the distribution of these receptors within the brain, which has been so far hampered by the lack of selective V1B markers. In the present study, we have determined the pharmacological properties of three new potent rat V1B fluorescent ligands and demonstrated that they constitute valuable tools for simultaneous visualization and activation of native V1B receptors in living rat brain tissue. Thus, d[Leu4,Lys-Alexa 647)8]VP (analogue 3), the compound with the best affinity-selectivity/fluorescence ratio for the V1B receptor emerged as the most promising. The rat brain regions most concerned by stress such as hippocampus, olfactory bulbs, cortex and amygdala display the highest V1B fluorescent labelling with analogue 3. In the hippocampus CA2, V1B receptors are located on glutamatergic, not GABAergic neurones, and are absent from astrocytes. Using AVP-EGFP rats, we demonstrate the presence of V1B autoreceptors on AVP-secreting neurones not only in the hypothalamus, but also sparsely in the hippocampus. Finally, using both electrophysiology and visualization of ERK phosphorylation, we show analogue 3-induced activation of the V1B receptor in situ. This will help to analyse expression and functionality of V1B receptors in the brain and contribute to further explore the AVPergic circuitry in normal and pathological conditions.

The impact of ß-azido(or 1-piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues.

The synthesis of new dermorphin analogues is described. The (R)-alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α-methyl-ß-azidoalanine or α-benzyl-ß-azido(1-piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [(3) H]DAMGO (a µ ligand) and [(3) H]DELT (a δ ligand). The most active analogue in this series, Tyr-(R)-Ala-(R)-α-benzyl-ß-azidoAla-Gly-Tyr-Pro-Ser-NH2 and its epimer were analysed by (1) H and (13) C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C(α) in the α-benzyl-ß-azidoAla residue in position 3. The (R) configuration led to the formation of a type I ß-turn, whilst switching to the (S) configuration gave rise to an inverse ß-turn of type I', followed by the formation of a very short ß-sheet. The selectivity of Tyr-(R)-Ala-(R) and (S)-α-benzyl-ß-azidoAla-Gly-Tyr-Pro-Ser-NH2 was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis, binding affinities and metabolic stability of dimeric dermorphin analogs modified with ß3-homo-amino acids.

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding ß(3)-homo-amino acids. The potency and selectivity of hybrid α/ß dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a µ ligand) and [3H]DELT (a δ ligand). Tha analog containing ß(3)-homo-Tyr in place of Tyr (Tyr-D-Ala-Phe-Gly-ß(3)-homo-Tyr-NH-)2 showed good µ receptor affinity and selectivity (IC50 = 0.302, IC50 ratio µ/δ = 68) and enzymatic stability in human plasma.

Synthesis, Biological Activity, and NMR-Based Structural Studies of Deltorphin I Analogs Modified in Message Domain with a New α,α-Disubstituted Glycines.

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)-α-benzyl-ß-azidoalanine, α-benzyl-ß-(1-pyrrolidinyl)alanine, α-benzyl-ß-(1-piperidinyl)alanine, and α-benzyl-ß-(4-morpholinyl)-alanine residues. The potency and selectivity of the new analogs were evaluated by a competitive receptor binding assay in the rat brain using [(3) H]DAMGO (a µ ligand) and [(3) H]DELT (a δ ligand). The affinity of analogs containing (R) or (S)-α-benzyl-ß-azidoalanine in position 3 to δ-receptors strongly depended on the chirality of the α,α-disubstituted residue. The conformational behavior of peptides modified with (R) or (S)-α-benzyl-ß-(1-piperidinyl)Ala, which displays the opposite selectivity, was analyzed by (1) H and (13) C NMR. The µ-selective Tyr-d-Ala-(R)-α-benzyl-ß-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 lacks the helical conformation observed in the δ-selective Tyr-d-Ala-(S)-α-benzyl-ß-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 . Our results support the proposal that differences between δ- and µ-selective opioid peptides are attributable to the presence or absence of a spatial overlap between the N-terminal message domain and the C-terminal address domain.

Biphalin analogs containing ß(3)-homo-amino acids at the 4,4' positions: Synthesis and opioid activity profiles.

Biphalin, a synthetic opioid octapeptide with a palindromic sequence has high analgesic activity. Biphalin displays a strong affinity for µ and δ-opioid receptors, and a significant to κ-receptor. The paper reports the synthesis of novel analogs of biphalin containing ß(3)-homo-amino acid residues at the 4,4' positions and a hydrazine or 1,2-phenylenediamine linker. The potency and selectivity of the peptides were evaluated by a competitive receptor-binding assay in rat brain homogenate using [(3)H]DAMGO (a µ ligand) and [(3)H]DELT (a δ ligand). Analogs with ß(3)-h-p-NO2Phe in positions 4 and 4' are the most active compounds. Selectivity depends on the degree of freedom between the two pharmacophore moieties. Analogs with a hydrazine linker show noticeable binding selectivity to µ receptors (IC50(µ)=0.72nM; IC50(δ)=4.66nM), while the peptides with a 1,2-phenylenediamine linker show slight δ selectivity (IC50(µ)=10.97nM; IC50(δ)=1.99nM). Tyr-d-Ala-Gly-ß(3)-h-p-NO2PheNHNH-ß(3)-h-p-NO2Phe (1) and (Tyr-d-Ala-Gly-ß(3)-h-p-NO2PheNH)2 (2) produced greater antinociceptive effect compared to morphine after i.t. administration.

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity.

New analogues of deltorphin I (DT I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 ), with the D-Ala residue in position 2 replaced by α-methyl-ß-azido(amino, 1-pyrrolidinyl, 1-piperidinyl or 4-morpholinyl)alanine, were synthesized by a combination of solid-phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to µ- and δ-opioid receptors. The affinity of analogues containing (R) or (S)-α-methyl-ß-azidoalanine in position 2 to δ-receptors strongly depended on the chirality of the α,α-disubstituted residue. Peptide II, containing (S)-α-methyl-ß-azidoalanine in position 2, displayed excellent δ-receptor selectivity with its δ-receptor affinity being only three times lower than that of DT I.

The biological consequences of replacing D-Ala in biphalin with amphiphilic α-alkylserines.

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, µ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α-alkylserines in position 2 and 2'. The incorporation of bulky α,α-disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N-migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor-binding assay in the rat brain using [(3)H]DAMGO (a µ ligand) and [(3)H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α-alkylserine, as analogues containing (R)-α-alkylserines displayed higher µ receptor affinity and selectivity than those incorporating the (S)-isomers.

Synthesis and receptor binding of opioid peptide analogues containing beta3-homo-amino acids.

beta-Amino acids containing hybrid peptides and beta-peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the micro- and delta-opioid receptors of beta-peptides, analogues of Leu-enkephalin, deltorphin I, dermorphin and alpha,beta-hybrides, analogues of deltorphin I. Substitution of alpha-amino acid residues with beta(3)-homo-amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation beta(3)h-D-Ala in position 2 or beta(3)hPhe in position 3 of deltorphin I resulted in potent and selective ligand for delta-opioid receptor. The NMR studies of beta-deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected.

Chemistry of alpha-hydroxymethylserine: problems and solutions.

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.

tert-Butyl N-[(S)-3-isopropyl-2-oxo-oxetan-3-yl]carbamate.

The structure of the title compound, C(11)H(19)NO(4), contains two crystallographically independent mol-ecules in the asymmetric unit. Both adopt the same conformation and they form pseudosymmetric R(2) (2)(8) dimers via two N-H⋯O hydrogen bonds. The dimers are linked by weak C-H⋯O inter-actions and are stacked in columns along the a axis.

tert-Butyl N-[(S)-3-isobutyl-2-oxooxetan-3-yl]carbamate.

The structure of the title compound, C(12)H(21)NO(4), contains two crystallographically independent mol-ecules in the asymmetric unit. Mol-ecules are linked into pseudosymmetric R(2) (2)(8) dimers through two N-H⋯O hydrogen bonds. The dimers are connected by weak C-H⋯O inter-actions, resulting in a three-dimensional network.

Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity.

In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by alpha-hydroxymethylserine (HmSer) and both enantiomers of alpha-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine beta-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine beta-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated epsilon-NH2 group of the inhibitor P1 position and beta-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.